Toxicity of aromatic solvents to microorganisms presents a major problem to work in the field of microbiology. Varied and poorly understood factors appear to influence solvent tolerance. Increasingly, attention has turned to genetic manipulation to create microbes that thrive in high concentrations of organic solvents. Understanding the mechanisms of solvent tolerance can be exploited in the future to generate microbes with enhanced biocatalytic potential.
One enzymatic pathway of increasing commercial interest controls toluene degradation. The first enzyme in the toluene degradation (TMO) pathway is toluene-4-monooxygenase (TMO; EC 1.14.13 and EC 1.18.1.3). Bacteria that possess the TMO pathway are useful for the degradation of toluene and other organics and are able to use these as their sole source of carbon (Wright et al., Appl. Environ. Microbiol. 60:235-242 (1994); Duetz et al., Appl. Environ. Microbiol. 60:2858-2863 (1994); Leahy et al., Appl. Environ. Microbiol. 62:825-833 (1996)). Bacteria that possess the TMO pathway are primarily restricted to the genus Pseudomonas. Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas aeruginosa and Pseudomonas mendocina are the most commonly utilized species.
Recently, various strains of Pseudomonas possessing the TMO pathway have been used to produce muconic acid from toluene via manipulation of growth conditions (U.S. Pat. No. 4,657,863; U.S. Pat. No. 4,968,612). Additionally, strains of Enterobacter with the ability to convert p-cresol to p-hydroxybenzoic acid (PHBA) have been isolated from soil (JP 05328981). Further, JP 05336980 and JP 05336979 disclose isolated strains of Pseudomonas putida with the ability to produce PHBA from p-cresol.
Although the above cited methods are useful for the production of PHBA, these methods are limited by the high cost and toxicity of the aromatic substrate, p-cresol. Furthermore, the above methods use an isolated wildtype organism that converts part of the p-cresol to PHBA while the remainder is further metabolized. The utility of these methods is limited by low yields and an inability to control further degradation of the desired product.
Previous studies indicated that cell growth and the activity of the enzymes in the metabolic toluene degradation pathway were inhibited in the presence of high concentrations of PHBA. Therefore, one problem to overcome is to develop a method of improving cell tolerance to high levels of PHBA or other aromatic solvents. During evolution, bacteria have developed a number of mechanisms which help to protect them from environmental toxins and various antibiotics. (Asako et al., Appl. Environ. Microbiol. 63:1428-1433 (1997); Aono et al., Appl. Environ. Miobiol. 60:4624-4626 (1994)). Many cytoplasmic membrane transport systems have been demonstrated to play an important role in bacteria by conferring resistance to toxic compounds. One of the most widespread is the active efflux of the toxic compounds from cells (Paulsen. et al., Microbiol. Rev. 60:575-608 (1996)).
Overexpression of an efflux system or its expression from a plasmid vector results in increased resistance of bacteria to a variety of toxic substances, while inactivation of an efflux system causes an increase in sensitivity to antibiotics and toxic substances (Li et al., J. Bacteriol. 180:2987-2991(1998); Ramos. et al., J. Bacteriol. 180:3323-3329 (1998)). Such efflux systems are increasingly being recognized in a wide range of bacteria. Comparative amino acid sequence analysis of various transport proteins plus function assays has enabled the identification of a number of distinct families and super-families of transports.